CN102387810A

CN102387810A - Identification of extracellular form of PTEN that can be used to treat tumors

Info

Publication number: CN102387810A
Application number: CN2010800163333A
Authority: CN
Inventors: 雷蒙·帕森斯
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2009-02-17
Filing date: 2010-02-17
Publication date: 2012-03-21
Anticipated expiration: 2030-02-17
Also published as: DK2400973T3; CA2752590C; HUE032028T2; BRPI1005951A2; EP2400973A4; EP2400973B1; JP2012517827A; CY1118861T1; PL2400973T3; JP2015231372A; US20120039861A1; JP5973035B2; WO2010096173A2; AU2016231517A1; PT2400973T; WO2010096173A3; AU2010216370A1; ES2623393T3; MX2011008695A; US9017981B2

Abstract

An isolated human phosphatase and tensin homolog long polypeptide (PTEN-long) comprising SEQ ID NO:1, fragments and analogues thereof, nucleic acids encoding such and compositions comprising such are provided. Methods to inhibit angiogenesis in a solid tumor, treat a solid tumor, and inhibit growth of a solid tumor using PTEN-long, fragments and analogues thereof, are provided.

Description

Discriminating can be used for treating the extracellular form of the PTEN of tumor

Related application

The application requires the priority of No. the 61/207th, 974, the U.S. Provisional Application case of 17 applications February in 2009, and the content of said patent is incorporated herein by reference.

Work disclosed herein is under government supports, under the CA082783 subsidy of National Cancer Institute (National Cancer Institute), to carry out.Therefore, U.S. government has some right to the present invention.

In the application's case, various publications are quoted with first author and time in bracket.Complete the quoting of these lists of references can be found in the ending of the description that is next to claims the place ahead.The mode that the disclosure of these open cases is quoted is in full incorporated in the application's case more fully to describe the prior art relevant with the present invention.

Technical field

Do not have

Background technology

PTEN TIF (PTEN tumor suppressor) is (referring to WO98/34624; Its mode of quoting in full is incorporated herein) be a kind of Cytoplasm phosphatase; It makes important second message,second messenger's

phosphatidylinositols

3,4, and (phosphatidylinositol 3 for 5-triphosphoric acid dephosphorylation; 4,5-triphosphate) (Ma Hama (Maehama) and Dixon (Dixon) 1998).This active downward modulation comprises anti-apoptotic path (apoptotic pathway), cell cycle progression and increase cellular metabolism (Li Shi (Sulis) of Soviet Union and Parsons (Parsons) 2003) by the many carcinogenic signal of the PIP3 activation initiation of Akt.The effect of PTEN in cancer lost through be everlasting heredity forfeiture or function many different tumor types from it, and obvious (Bang Nu (Bonneau) and grand base (Longy) 2000).It lacks initial discovery in the neuroglia cancer, and after this discovery takes place relevant with the tumor of prostate, breast, endometrium, melanocyte, kidney and lung again.The system genitale sudden change of PTEN is also relevant with genetic cancer body constitution syndrome, steps on syndrome (Cowden ' s Syndrome) (English lattice (Eng) 2003) such as examining.The mouse model of PTEN forfeiture has been recurred it in heterozygote mice and tissue-specific gene rejecting person (tissue specific knockout); In many histological types as effect (all promises of enlightening Christo (Di Cristofano), the Pei Sai people 1998 such as (Pesce) of TIF; Overstate than-A Duoing (Kwabi-Addo) lucky people 2001 such as (Giri); Pendant Qu Xili (Petrocelli) and this woods Crane (Slingerland) 2001; Especially (You), people 2002 such as (Castrillon) is swelled in the Castries; Fu Leize (Fraser), people 2004 such as (Zhu) Zhu).

Pten protein contains N end dual specificity phosphatase enzymatic structure territory (N-terminal dual specificity phosphatase domain) and C end C2 phospholipids incorporate domain (C-terminal C2 phospholipid binding domain); Subsequently for exist phosphorylation site to have not structuring afterbody (Lee (Lee), the poplar people 1999 such as (Yang) of regulation and control importance because of inside; Watt Fran Vazquez (Vazquez) draws Maas watt rice people 2000 such as (Ramaswamy); Toure this (Torres) and Pu Lidu (Pulido) 2001; Watt Fran Vazquez (Vazquez), Groceman people 2001 such as (Grossman)).Pten protein mainly is present in the Cytoplasm, yet, there is more and more evidences proof PTEN to be present in the nucleus, this location makes protein list ubiquitinization (monoubiquitination) regulate and control (Bake (Baker) 2007 through NEDD4-1; King (Wang), Julia Trotman people 2007 such as (Trotman)).

Ribosome scans 5 ' UTR before translation initiation takes place at start codon (start codon) AUG place.Although ribosome is understood with the practical ways that decides suitable start codon yet fully, there is some character of indicating preinitiation complex (pre-initiation complex) to slow down its scanning wherein and begin to translate in mRNA itself with sequence.Shown classics " V sequence (Kozak sequence) " CCACC ATGG (wherein underlined ATG is start codon (initiation codon)) is to help initial sequence situation (sequence context) (V (Kozak) 1991) most.The mRNA secondary structure also possibly promote initial through the scanning of the actual preinitiation complex that slows down, because scanning needs helicase to untie secondary structure (V ((Kozak)) 1990) before reading.

In some transcript, translation initiation can begin to carry out from non-AUG codon.This less percentage ratio and result who only comprises usually from the gross protein that the transcript translation comes produces the different proteinic compounding substances of N end.V (Kozak) is described from the efficient of the initial translation of non-AUG codon and is found that GUG and CUG can both initially translate in vitro, but efficient much lower (V (Kozak) 1989).Further research shows; The availability of methionine can change the confusion of translation initiation through unclear mechanism still; To make eIF2 phosphorylation, eIF2 by nutrient sensitivity kinases (nutrient sensitive kinase) be that ((Hershey) 1991 wished in the Hull for the component of 43S preinitiation complex but possibly relate to; (Hann) 1994 received in the Chinese).

Shown that numerous protein begins translation from substituting start codon (alternate initiation codon).Transcription factor (transcription factor) c-myc has substituting upper reaches CUG start codon, and when translating, it adds 14 aminoacid (Chinese is received (Hann) and graceful (Eisenman) 1984 in Essen) at proteinic N end.Shown this substituting with merit iso series (alternate isoform) being selected property destruction (Chinese is received (Hann), gold people 1988 such as (King)) in burkitt's lymphoma (Burkitt ' s lymphoma).In tissue culture, when methionine was in low concentration, the myc of more microscler formula mainly transcribed (Chinese is received (Hann), Suo Luoan Blang people 1992 such as (Sloan-Brown)) under high-cell density.Further research discloses, and the c-myc of more microscler formula has growth inhibited property and has one group of different with classical c-myc albumen target (Chinese is received (Hann), Di Kete people 1994 such as (Dixit)) of transcribing.(Fu Luojiweizi (Florkiewicz) and Sa Mo (Sommer) 1989) (pula thatch (Prats), Ka Gede people 1989 such as (Kaghad)).

In addition, the actual Subcellular Localization of known protein matter can be by substituting start codon decision.At mice proto-oncogene (proto-oncogene) int-2 from the upper reaches under the substituting initial situation; CUG codon Codocyte is appraised and decided the position; And AUG codon coding is used to be positioned to secrete the signal peptide (signal peptide) (A Kelande (Acland), Dixon people 1990 such as (Dixon)) of path (secretory pathway).Similar phenomenon is described in human FGF3, wherein be appointed as the secretion path from the protein of AUG translation, and the protein positioning that CUG translates from the upper reaches is in nucleus (Ji Fu (Kiefer), A Kelande people 1994 such as (Acland)).In addition, in such as some eukaryotic proteins such as TEF-1 and PRPS-3, protein is fully from initial (flat (the Taira) ， Fan Bamboo grass people 1990 such as (Iizasa) of CUG codon; Xiao (Xiao), Dai Weidesen people 1991 such as (Davidson)).

Be appointed as the one section hydrophobic amino acid targeting endoplasmic reticulum (endoplasmic reticulum) (Blobel (Blobel), Wal special people 1979 such as (Walters)) of protein through being called signal peptide of secretion (secretion).Usually the signal peptide that is present in proteinic N end when translation binding signal identification granule (signal recognition particle SRP) and ribosome is suspended and translocate to ergastoplasm, combines the SRP receptor there.In case the ribosome butt joint, the SRP-SRP receptor complex is released, and translates through Sec61 translocon (translocon) from newly beginning to pass the inner chamber of ER.After soluble protein discharged protein from the Sec translocon, signal peptide is cracking subsequently.Cross at protein under the situation of cell thin film (membrane), transbilayer helix (transmembrane helix) serves as the signal peptide of ER transposition.These protein in Golgi body (golgi) widely through glycosylation modified and shuttle back and forth to plasma membrane (plasma membrane) (Alberta (Alberts) 2002) with the excretion vesicles form.

It is very important for cancer to have shown many secreted proteins (secreted protein).For example, shown that Wnt signal conducting path (signaling pathway) can change in pulmonary carcinoma.Wnt is a kind of secreted part (secreted ligand) of FZ (Frizzled) receptor family.The Wnt activation of FZ makes albumen at random (disheveled) white (the degraded complex of β-catenin) of beta-catenin that dissociates; It comprises APC; Thereby make the white content rising of beta-catenin and translocate to nucleus, it can interact and trans activation (transactivate) TCF transcription factor there.Activation sudden change during passivation sudden change among the APC and beta-catenin are white is specified in heritability and sporadic colon cancer.In addition, many extracellular ligand antagonisies (extracellular ligand antagonist) (such as SFRP and Wnt-5a) and Wnt competing phase FZ receptor together.The both has shown it is TIF; The SFRP gene knockout mice develops and lymphoma, and in melanoma, has detected the outer genetic silencing (epigenetic silencing) of Wnt-5a.

Open report all about PTEN points out that all protein positioning is in Cytoplasm or nucleus.Find that many extracellular proteins (glypican (glypican) and syndecan (syndecan)) combine PTEN, related numerous protein (reticulocalbin protein (reticulocalbin) and calcium chamber albumen (calumenin)) also combines PTEN in the secretion path.Suppose that PTEN gets into the secretion path, thereby can carry out said interaction.In fact, as disclosed herein, have the protein of novel difference translation, be called PTEN-long, it contains N end signal peptide and it is by cell exocrine.

Summary of the invention

A kind of separated human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprises continuous amino acid residue with sequence shown in the SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5 or its variant separately.

One peptide species, it comprises the residue 1 to 173 of (i) SEQ ID NO:1, or (ii) comprises its analog of the residue 1 to 173 of SEQ ID NO:5, or the (iii) residue 22 to 516 of SEQ ID NO:1.

A kind of pharmaceutical composition; It contains phosphatase and the long polypeptide of tensin congener (PTEN-long) that comprises the continuous amino acid residue; Said amino acid residue has sequence shown in the SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5.

A kind of method of treating experimenter's entity tumor; It comprises and gives said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) with continuous amino acid residue of sequence shown in the SEQ ID NO:1 that comprise; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with effective treatment experimenter's entity tumor.

A kind of method of growth of the entity tumor that suppresses the experimenter; It comprises and gives said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) with continuous amino acid residue of sequence shown in the SEQ ID NO:1 that comprise; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with effective growth that suppresses experimenter's entity tumor.

The method of the angiogenesis in a kind of entity tumor that suppresses the experimenter; It comprises and gives said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) with continuous amino acid residue of sequence shown in the SEQ ID NO:1 that comprise; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with the angiogenesis in effective inhibition experimenter's the entity tumor.

Apoptotic method takes place in the blood vessel epithelial cell of the blood vessel in a kind of experimenter's of inducing the entity tumor; It comprises and gives said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) with continuous amino acid residue of sequence shown in the SEQ ID NO:1 that comprise; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with the blood vessel epithelial cell generation apoptosis of the blood vessel in the entity tumor of effectively inducing the experimenter.

A kind of method of treating experimenter's entity tumor; It comprises and gives said experimenter a certain amount of expression vector; Said expression vector codes comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) of SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog of SEQ ID NO:5, in the cell of entity tumor, to express effectively PTEN-long, PTEN-long fragment or its analog of the amount of the said experimenter's of treatment entity tumor.

A kind of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, it is used to treat experimenter's entity tumor, suppresses experimenter's entity tumor growth; Induce the blood vessel epithelial cell generation apoptosis in experimenter's the entity tumor, or suppress the angiogenesis in experimenter's the entity tumor.

A kind of compositions; It comprises the human phosphatase enzyme that SEQ ID NO:1 and the long polypeptide of tensin congener (PTEN-long) of the non-peptide reagent of bonding (non-peptide agent); Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, said non-peptide reagent increases the plasma half-life (plasma half-life) of PTEN-long, PTEN-long fragment or its analog respectively.

A kind of separated nucleic acid; Its coding comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) of SEQ ID NO:1; Or coding comprises its fragment of residue 22 to 516 of SEQ ID NO:1, or coding comprises its analog of SEQ ID NO:5.

A kind of expression vector (expression vector); It comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that coding comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise the nucleic acid of its analog of SEQ ID NO:5.

A kind of transformant (trans formed cell); It can express human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, will encode the recombinant DNA of PTEN-long or its analog of wherein said cell is incorporated in its genome.

A kind of host cell (host cell); It can express human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, wherein said host cell comprises the plasmid of coding PTEN-long or its analog.

A kind of method, it comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that mixing (1) comprises SEQ ID NO:1, or comprises its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprises its analog of SEQ ID NO:5; (2) the non-peptide reagent of the plasma half-life of increase PTEN-long or its analog; So that comprise the PTEN-long of SEQ ID NO:1 or comprise SEQ ID NO:1 residue 22 to 516 its fragment or comprise its analogue bonded non-peptide reagent of SEQ ID NO:5, said non-peptide reagent increases the plasma half-life of PTEN-long, PTEN-long fragment or its analog respectively.

A kind of separated nucleic acid molecules, it is formed with the complementary series of SEQ ID NO:2 or SEQ ID NO:6 nucleotide sequence or with its complementary series (complement) of SEQ ID NO:2 or SEQ ID NO:6 specific hybrid respectively by the fragment with at least 20 nucleotide of SEQ ID NO:2 or SEQ ID NO:6 nucleotide sequence or under stringent condition.

A kind of separated antibody or its fragment, wherein said antibody or its fragment combine (1) to comprise the PTEN-long polypeptide of the amino acid residue 1-173 of SEQ ID NO:1; Or (2) comprise its analog of SEQ ID NO:5; Or (3) have the aminoacid of sequence shown in the SEQ ID NO:3; Or (4) comprise the PTEN-long fragment of the residue 22 to 516 of SEQ ID NO:1.

A kind of separated antibody or its fragment; Wherein said antibody or its fragment combine the conformation epitope (conformational epitope) of PTEN-long; Wherein said PTEN-long comprises the sequence shown in the residue 1-173 of SEQ ID NO:1, but wherein said antibody does not combine PTEN.

A kind of separated antibody or its fragment, wherein said antibody or its fragment combine the epitope of PTEN-long, and wherein said PTEN-long comprises the sequence shown in the residue 1-173 of SEQ ID NO:1, but wherein said antibody does not combine PTEN.

It is active that the fragments of peptides of a kind of SEQ ID NO:1 or SEQ ID NO:5, said fragment have antitumor, angiogenesis inhibitor or anti-apoptotic.

Description of drawings

Fig. 1 .PTEN-long constructs the figure of body (Construct).Show to form and to be used for driving the expression that begins to express the combination of PTEN from endogenous initiation site (endogenous start site) or substituting initiation site (alternate start site) and to construct body.Classical PTEN is with black display, and the translation area among the UTR shows with Lycoperdon polymorphum Vitt.

Fig. 2. the figure of homo sapiens (Homo sapiens) PTEN mRNA.PTEN mRNA coding with shown in classical ATG start codon with frame and be positioned at 173 aminoacid at its upper reaches.Translation begins from the CTG of the nucleotide-519 at the classical ATG upper reaches.Prolongation is shown as the residue 1-173 of SEQ ID NO:1.

The comparison (Alignment) of the N end of the lineal congener of Fig. 3 .PTEN.Go up use BLOSUM62 score matrix (scorematrix) comparison from the pten protein sequence of specifying species at Vector NTI (hero company (Invitrogen)).The N terminal sequence of the prolongation of homo sapiens and house mouse (Mus musculus) (Ah's Hilde Strike this (asterix)) uses ORFinder (NCBI); From disclosed mRNA, the classical AUG start codon of service range is the substituting start codon translation of CUG that-519 (homo sapiens) and-520 (house mouses) are located.From homo sapiens's mRNA sequence (NM_000314) with from the mRNA sequence (NM_008960) of house mouse.Apis mellifera Linnaeus (Apis mellifera) sequence is to obtain from Baylor College Medicine Apis genome plan (Baylor College of Medicine Honey Bee Genome Proj ect).The protein sequence (Daf-18) of Caenorhabditis elegans (Caenorhabditis elegans) PTEN is to download from Wormbase.Cattle (Bos Taurus) (XM_613125) and chimpanzee (Pan troglodytes) be to download (XP_521544) from NCBI.

Fig. 4. there is the evidence of PTEN-long.A) investigation has the different cell lines of two kinds of different PTEN antibody.MCF10A and HEK293 are the wild types of PTEN.BT549 and HCC1937 are that PTEN deletion form (PTEN null) and ZR-75-1 locate to have sudden change at PTEN (L136); B) further investigate different cell lines with identification PTEN and PTEN monoclonal antibody of PTEN-long; C) a large amount of PTEN-long of Wt ES cellular expression.PTEN-long expresses responsive to the stable PTEN shRNA in these cells and is not present in fully in the PTEN gene knockout cell.PAkt content is relevant with the PTEN content back to a great extent; D) expression of PTEN and PTEN-long in the feasible blocking-up of the PTEN siRNA HEK293 cell.E) exogenous expression of plasmid in PTEN deletion form PC3 cell line.The ORF (the 2nd swimming lane) that PTENorf only encodes and begins from start codon AUG.Add ATR (the substituting translation area of ATR=) and make it possible to faintly translate PTEN-long (the 3rd swimming lane).Upper reaches initiation site sports ATG makes the protein complement change PTEN-long (the 5th and the 6th swimming lane) fully into.The ATG start codon sports ATA and has eliminated 55kDa band (the 4th and the 6th swimming lane).F) to the antibody of PTEN deletion form U87 cell line of plasmid (ATG/ATG) that produces and be used for the molten born of the same parents' thing (cell lysate) of cell and the overexpression PTENorf or the 5 ' ATR that encodes of HEK293 by 5 ' ATR amino acids coding.PTEN-long is found in the cell of overexpression 5 ' ATR only.Viewed background band is present in trace (blot) bottom in the U87 cell.

Fig. 5. signal peptide prediction.Among translation PTEN 5 ' UTR sequence and the input SignalIP3.0.Use the hidden Markov model (Hidden markov model) of eukaryotic signal peptide to predict.The positively charged N terminal sequence of N district (N-region) expression signal peptide.The H district is the hydrophobicity core of signal peptide.The C district is that proline destroys the spiral of hydrophobicity core usually by the slight polarity zone of proline labelling.Cracking probability (cleavage probability) thus indication cracking site release signal peptide allows protein to be discharged in the ER inner chamber.(wearing shellfish (Dalbey) and dark fund (Heijne), 2002).Cracking takes place in 21 places in the position in prediction.

Fig. 6. concanavalin A (ConcanavalinA) is caught (pulldown).Dissolving HEK293 cell and use Con A Concanavalin agarose are caught glycosylated protein.Decompose eluent and carry out immunoblotting through SDS-PAGE to PTEN (6H2.1).With respect to input quantity, can in catching thing, observe the PTEN-long enrichment.Notice the enrichment of longer PTEN band.PTEN has a plurality of possible O-glycosylation sites (O-glycosylation site), but only has a N-glycosylation site.Whether we use a part that agglutinin Con A Concanavalin-A (lectin concanavalin-A) of combining sugar moieties confirms PTEN complement in the HEK293 cell by glycosylation in catch analyzing.We can purification contain the PTEN mixture of about 50%PTEN-long, and wherein PTEN-long is with respect to a large amount of enrichments of normal PTEN.This explanation PTEN-long is glycosylated and the Cytoplasm 55kDa form of PTEN is glycosylated or PTEN-long cracking in the extracellular.

Fig. 7 .PTEN and PTEN-β combine heparan (heparan).Make the Mouse Liver extract through 1ml HiTrap heparan agarose (Heparan sepharose) (peace agate West Asia company (Amersham)) post.Wash said post and with the 1M NaCl elute protein of continuous column volume with 500mM NaCl.Use the PTEN monoclonal antibody to analyze the PTEN in the fraction (Fraction) through SDS-PAGE.The previous PTEN that shown has affinity, a kind of character (Da Si (Das), Dixon people 2003 such as (Dixon)) that causes the high anionic property PtdIns of PTEN preference (3,4,5) P3 for the material of band height negative electricity.Because heparan is one of biomolecule with maximum negative electricity, so we suppose that heparan effectively mediates combining of PTEN and extracellular matrix (extracellular matrix).Use is from the protein extract of mouse liver, and we find that PTEN combines heparan with high-affinity.In addition, from use 1M NaCl from the heparin-agarose post continuously eluting PTEN also eluting go out PTEN-long.

Fig. 8. the protease protection is analyzed.With HEK293 cell resuspending in the E.C. 3.4.21.64 (proteinase K) of progressive concentration.Triton is added in the reaction of the E.C. 3.4.21.64 that contains maximum concentration to ultimate density be 0.2%.Prepare cell with the PMSF stopped reaction and in La Muli buffer (laemlli buffer) and dissolve born of the same parents' thing.On 8% PAAG, resolve molten born of the same parents' thing and carry out immunoblotting through SDS-PAGE to PTEN (6H2.1), AKT, E-cadherins (E-cadherin).The PTEN immunoblotting represent PTEN-long than big band.These data show E-cadherins and PTEN-long mainly are positioned on the cell surface.

Fig. 9. from the PTEN of the heparin affinity purification of conditioned medium and the high eluting salt of PTEN-long.Can be from HiTrap heparin (peace agate West Asia company (Amersham)) post eluting PTEN and PTEN-long after carrying out affinity purification from conditioned medium.To the monoclonal antibody of PTEN afterbody (top) with have specific antibody and all discern the protein band of quality translating aminoacid among 5 ' ATR for about 55kDa.

Figure 10. from human serum purification PTEN.With a-protein/G pre cleaning from the antibody in the human serum of AB blood and through heparin-agarose.Carry out immunoblotting or only use secondary antibody (secondary) to carry out polluting through SDS-PAGE parsing eluent and to PTEN with the control heavy chain.

The anti-angiogenesis activity of Figure 11 A-11C.PTEN-long.(A) express in the subclass of blood vessel and the blood capillary of PTEN-long in developmental retina.Effect during this expression pattern is degenerated with the classical field formalism formation sharp contrast of PTEN and with the blood vessel that PTEN-long takes place in inducing these zones is consistent.When the Western blotting (western blot) that dissolves born of the same parents' thing according to top-right full retina; Utilize hyperoxia (B) to induce this blood vessel degenerative process; And when inducing this blood vessel degenerative process through the PTEN-long in the endotheliocyte under hypoxia condition forfeiture (C), the remarkable rise of PTEN-long is enhanced this dependency.These discoveries show that PTEN-long is suitable for and make anti-angiogenic therapy, for example treat diabetic retinopathy and high hypertrophy property vascular disorder.Arrow indication CD34 and the positive tissue of PTEN-long (blood vessel).

The short apoptosis of Figure 12 .PTEN-long is active.As indicated, cell death inducing in 24 hours MCF-10A breast epithelial cell of purified PTEN-long processing.Casprotease 3 (Caspase 3) cracking indicator cells apoptosis activity.

Figure 13. handle mice with PTEN-long.Pass through the figure that caliper (Caliper) is measured measured tumor size after ten days with PTEN-long or empty carrier contrast (Empty Vector Control) processing.With the pcDNA3.1His V5 carrier rotaring redyeing 293 cell that contains ATG/ATG PTEN-long.48 hours preparation Cytoplasms dissolve born of the same parents' things and the V5-antibody beadlet and with V5 peptide eluting of flowing through after transfection.The Western blotting of V5-beadlet purification eluent is showed in the below.Original observed can be used for treating tumor to PTEN-long.The mammary fat pad of using U87 glioblastoma cells (glioblastoma cell) (100 ten thousand) to inject the scid mice is set up xenograft (Xenograft).After transplanting, begin in about two weeks to handle.

Figure 14. handle the result of mice with PTEN-long.Figure shows the survival branch rate (in the sky) with contrast and 14 days mice of PTEN-long injection processing.

Figure 15. the appointment that the G at PTEN (lack 5 ' UTR PTENorf), PTEN-long and aminoacid 305 places is sported the PTEN-long of R (it is similar to G129R sudden change among the PTENorf) is constructed the body transfection in 293 cells.Use show that PTEN-long is an active phosphatases, and PTEN-long G305R mutant (it is G129R in PTEN) reduces phosphatase activity from the purified protein of these cells.

Figure 16. in the experiment of using PTEN-long (G305R) mutant, show that phosphatase activity is that the PTEN-long activity is necessary.Based on the PTEN document, the known truncate of in the C2 domain, being done makes protein unstable, and based on the PTEN crystal structure, believes that the interaction between C2 domain and the phosphatase domain is most important for phosphatase activity.Therefore, at the C end, the active minimal structure of PTEN-long territory will need the C2 domain, but not need afterbody.At N end, the cracking site of prediction is at aminoacid 21 places, and therefore proteinic functional areas in this zone.With regard to this point, be important to note that when with PTEN or with PTEN-long parallel processing U87 tumor, PTEN handles the obvious effect of not observing, only PTEN-long handles has obvious effect.

Figure 17. from purification PTEN-long through 293 cells of the pcDNA3.1 expression vector transfection that contains ATG/ATG-PTEN long with His and V5 label.At Ni ⁺Behind the post eluting, in solvent resistant column, resolve eluent.OD280 shows with blue line.Be rich in PTEN-long among the fraction 7-18.The output of this experiment is about 1mg.The PTEN-long product of arrow indication PTEN-long and migration change.

Figure 18 A-18B. (A) LNCaP prostate gland cancer cell uses cell death as reading (protein is to carry out purification through ARVYS) to the purified proteinic dose response of PTEN-long (DoseResponse).1 * equal every milliliter 0.33 microgram.In the culture medium of no somatomedin, handle cell.After 24 hours, with serum-free medium washed cell and dissolving in La Muli sample buffer (Laemmli sample buffer).For specifying protein to carry out Western blotting.(B) the U87 glioblastoma cells of handling with PTEN-long, PTEN-long (G305R) or simulation contrast (Mock control) shows cell death inducing, and is indicated like the downward modulation of PARP cracking and serine 473 pAKT of place signals.But these data further confirm the PTEN-long cell death inducing and reduce the conduction of PI3K/AKT signal.

Figure 19 A-19C. can reduce tumor size as utilizing caliper and using luciferase reporter gene (luci ferase reporter) to combine true (xenogen) live animal imaging system of smart promise measured through the PTEN-long of AKTA purification albumen in five day time.Give every day with the about 0.05mg PTEN-long of mice and continue five days.100 ten thousand U87 glioblastoma cells are injected in the mammary fat pad of expressing luciferase, thereby set up xenograft owing to infect FUW-luciferase-neo.Forming images preceding 10 minutes with the true imaging system of smart promise, intraperitoneal is given injected in mice fluorescein (luciferin).(A) (left figure) and processing the 5th day (right figure) are carried out the luciferase measurement to 4 mices before processing.(B) before processing and during handling 5 days, carry out caliper and measure, with cm ²Meter.(C) the detected photon of the smart Nuo Zhen system of utilization as being formed images among the figure.The standard error of four mices in the demonstration group.The 0th day to the 5th day detected photon carried out student t check (Student t-test).

Figure 20. in independent experiment, let the U87 tumor growth to 1.5cm ², use PTEN (orf-403 aminoacid subsequently; N=5), PTEN-long (G305R; N=5) or wild type PTEN-long (n=4) handle.Handle after 5 days, the average luminescence of the mice of handling through PTEN-long changes (average change luminescence) and shows significantly and reduce, but does not reduce through the group that PTEN or PTEN-long (G305R) handle.Luminous minimizing reduces relevant with tumor size.These data declarations PTEN-long needs 5 ' ATR and phosphatase activity to work.

The U87 xenograft that Figure 21 A-21C. handles through PTEN-long analyze the inhibition that activation and the PI3K signal of clear-cells apoptosis conducts.Handled tumor as stated 5 days.(A) designated treatment is collected after 5 days through the tumor of PTEN-long wild type and G305R processing and is also dissolved to carry out western blot analysis (western analysis).The wild-type protein of PTEN-long can reduce FOXO and AKT phosphorylation and activation Casprotease-3 cracking.(B) will be fixed in the formalin (formalin) and use FFPE like 5 days representative tumor of specified processing.To the antibody that detects cracked Casprotease-3 (apoptotic label) with section statining.The apoptotic cell percentage ratio of the cell of handling through PTEN-long significantly increases P=0.0419, student t check.(C) the painted representative image of cracking Casprotease-3.

Figure 22 A-22B. is in 5 days identical tumor of PTEN-long processing, and number of blood vessel significantly reduces.Designated treatment is collected the tumor of handling through PTEN-long wild type and G305R and is fixed in the formalin and uses FFPE after 5 days.To the antibody that detects CD31 (as the label of the endotheliocyte of lining blood vessels) with section statining.(A) number of blood vessel in each visual field of the cell that PTEN-long handles significantly reduces (40 * object lens), P=0.007579, student t check.(B) the painted representative image of CD31.

Figure 23 A-23B. sets up the U87 xenograft in 6 groups (every group of n=3), and through (IT), subcutaneous (SC), intravenous (IV) injection in intramuscular (IM), intraperitoneal (IP), the tumor, handles four days with PTEN-long.(A) as utilizing the 0th day to the 4th day measured average tumor change in size (CM2) of caliper.(B) the representative image of the true imaging of smart promise is showed on the right side.From these data, our deducibility goes out with undressed mice to compare, and all injecting methods all can influence tumor growth, and only shows that through the group that IM handles amount of degradation is significantly lower.

Figure 24 A-24C. is to carrying out heteroplastic transplantation experiment (Xenograft experiment) from breast, brain and prostatic 6 cell lines.The top is the variation diagram that 4 breast cancer cell lines are drawn.(A, B and D) process designated treatment natural law, as utilize the measured tumor surface of caliper to amass (cm ²) figure.(C) processing is only promptly seen the variation in the HCT-1143 cell after 24 hours.In all four cell lines, the tumor size after the processing obviously reduces.

Figure 25 .PTEN-long is incorporated into cell.PTEN-long albumen added in U87 cell culture medium on ice continue 10 minutes, fixing, then dye with its antibody of identification to PTEN-long.

Figure 26. this analysis of mayer (Miles Assay): PTEN-long suppresses inducing of vascular permeability (vascular permeability).This inhibition can reverse through purified protein is cultivated with PTEN antibody (6H2.1) in advance.PTEN-long can suppress the inductive vascular permeability of VEGF.This inducing can be recovered through PTEN-long is cultivated with the antibody that produces to PTEN in advance, but can not recover with contrast IgG.

The specific embodiment

One peptide species, it comprises the residue 1 to 173 of (i) SEQ ID NO:1, or (ii) comprises its analog of the residue 1 to 173 of SEQ IDNO:5, or the (iii) residue 22 to 516 of SEQ ID NO:1.

A kind of pharmaceutical composition; It contains phosphatase and the long polypeptide of tensin congener (PTEN-long) that comprises the continuous amino acid residue; Said continuous amino acid residue has sequence shown in the SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5.

In an embodiment of methods described herein, tumor is a cancerous tumour.In an embodiment of methods described herein, cancerous tumour is the tumor of experimenter's neurogliocyte, prostate, ovary, uterus, endometrium, breast, melanocyte, kidney, lung, colon, head, cervical region or pancreas.

In an embodiment of methods described herein, cancerous tumour is by PTEN or by (PI3Kpathway) activation of PI3K path or be the PTEN feminine gender.

In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are through giving the experimenter in the direct introducing entity tumor.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are to be injected in the entity tumor.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are directly to introduce in the entity tumor through conduit.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are to give the experimenter in the blood vessel through direct introducing provision entity tumor.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are to be injected in the blood vessel of provision entity tumor.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are directly to introduce in the blood vessel of provision entity tumor through conduit.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are that intravenous gives the experimenter.In an embodiment of methods described herein, PTEN-long or its analog or PTEN-long fragment are the subcutaneous experimenters of giving.

A kind of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, it is used to treat experimenter's entity tumor, suppresses the growth of experimenter's entity tumor; Induce the blood vessel epithelial cell generation apoptosis of experimenter's entity tumor, or the angiogenesis in inhibition experimenter's the entity tumor.

At an embodiment of the chemical compound that is used for purposes described herein, tumor is a cancerous tumour.In an embodiment of methods described herein, cancerous tumour is neuroglia cancer, carcinoma of prostate, breast carcinoma, carcinoma of endometrium, melanocarcinoma, renal carcinoma or pulmonary carcinoma tumor.

At an embodiment of the chemical compound that is used for purposes described herein, cancerous tumour is by PTEN or by the activation of PI3K path or be the PTEN feminine gender.

A kind of compositions; It comprises the human phosphatase enzyme that SEQ ID NO:1 and the long polypeptide of tensin congener (PTEN-long) of the non-peptide reagent of bonding; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, said non-peptide medicament increases the plasma half-life of PTEN-long, PTEN-long fragment or its analog respectively.

In one embodiment, compositions described herein further comprises pharmaceutical carriers.In an embodiment of compositions described herein, the non-peptide reagent that increases plasma half-life is Polyethylene Glycol (PEG).In an embodiment of said compositions, the C of PEG bonding PTEN-long, PTEN-long fragment or its analog end or N end.In an embodiment of said compositions, the non-peptide reagent that increases plasma half-life is chloro-carbonic acid-9-fluorenyl methyl ester (Fmoc) or (7-sulfonic group)-9-fluorenylmethyloxycarbonyl.

In one embodiment, nucleic acid comprises the continuous nucleotide with sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.In one embodiment, nucleic acid comprises the continuous nucleotide residue 503 to 2243 of sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.In one embodiment, nucleic acid comprises the continuous nucleotide with sequence shown in SEQ ID NO:3 or the SEQ ID NO:7.In one embodiment, nucleic acid comprises the continuous nucleotide residue 503 to 2243 of sequence shown in SEQ ID NO:3 or the SEQ ID NO:7.In one embodiment, nucleic acid is RNA.In one embodiment, nucleic acid is DNA.In one embodiment, nucleic acid is cDNA.

A kind of expression vector; It comprises nucleic acid; Said nucleic acid coding comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) of SEQ ID NO:1, or comprises its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprises its analog of SEQ ID NO:5.

A kind of transformant; It can express human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, will encode the recombinant DNA of PTEN-long or its analog of wherein said cell is incorporated in its genome.

A kind of host cell; It can express human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, wherein said host cell comprises the plasmid of coding PTEN-long or its analog.

In one embodiment, host cell is a bacterial cell.In one embodiment, host cell is a mammalian cell.

A kind of method, it comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that mixing (1) comprises SEQ ID NO:1, or comprises its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprises its analog of SEQ ID NO:5; (2) the non-peptide reagent of the plasma half-life of increase PTEN-long or its analog; So that comprise the PTEN-long of SEQ ID NO:1 or comprise SEQ ID NO:1 residue 22 to 516 its fragment or comprise its analogue bonded non-peptide reagent of SEQ ID NO:5, said non-peptide reagent increases the plasma half-life of said PTEN-long, PTEN-long fragment or its analog respectively.

A kind of separated nucleic acid molecules, it is formed with the complementary series of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:6 or with its complementary series of SEQ ID NO:2SEQ ID NO:6 specific hybrid by the fragment with at least 20 nucleotide of SEQ ID NO:2 or SEQ ID NO:6 nucleotide sequence or under stringent condition.

In one embodiment, antibody is monoclonal antibody (monoclonal antibody).In one embodiment, antibody is antibody fragment.In one embodiment, antibody fragment is Fab, Fab ', F (ab ') ₂Or Fv fragment.In one embodiment, antibody fragment is single-chain antibody (single-chain antibody).In one embodiment, antibody is humanized antibody (humanized antibody).

A kind of separated antibody or its fragment; Wherein said antibody or its fragment combine the conformation epitope (conformational epitope) of PTEN-long; Wherein said PTEN-long comprises the sequence shown in the residue 1-173 of SEQ ID NO:1, but wherein antibody does not combine PTEN.

In one embodiment, antibodies comprises any one the epitope (epitope) among the residue 153-173 of SEQ ID NO:1.

A kind of separated antibody or its fragment, wherein said antibody or its fragment combine the epitope of PTEN-long, and wherein said PTEN-long comprises the sequence shown in the residue 1-173 of SEQ ID NO:1, but wherein antibody does not combine PTEN.

In one embodiment, the residue 153-173 of antibodies SEQ ID NO:1 or its part.

It is active that the fragments of peptides of a kind of SEQ ID NO:1 or SEQ ID NO:5, said fragment have antitumor, angiogenesis inhibitor or anti-apoptotic.In one embodiment, peptide comprises 5-10,10-20 is individual, 20-30 is individual or 30-40 aminoacid.In one embodiment, peptide comprises the amino acid/11-173 of SEQ ID NO:1 or SEQ ID NO:5.In one embodiment, peptide comprises the continuous amino acid residue 22 to 516 of SEQ ID NO:1.In one embodiment, peptide does not comprise the continuous amino acid residue 174 to 576 of SEQ ID NO:1 or SEQ ID NO:5.

As used herein, " control effectively (prophylactically effective) " of material amount is that effectively prevention or postpone gives the amount that the experimenter's of said material particular pathologies condition of illness (given pathological condition) shows effect.

As used herein, " treatment effectively (therapeutically effective) " of material amount is effective treatment, improve or alleviate symptom or the amount of the cause of disease of the experimenter's who suffers from particular pathologies condition of illness and give said material particular pathologies condition of illness.

In one embodiment, treatment or control effective dose are that the about 1mg reagent of every experimenter of each administration is to each about 1g reagent of every experimenter of administration.In another embodiment, treatment or control effective dose are every about 10mg reagent of experimenter to every experimenter 500mg reagent.In another embodiment, treatment or control effective dose are every about 50mg reagent of experimenter to every experimenter 200mg reagent.In another embodiment, treatment or control effective dose are every about 100mg reagent of experimenter.In another embodiment, treatment or control effective dose are to be selected from every experimenter 50mg reagent, every experimenter 100mg reagent, every experimenter 150mg reagent, every experimenter 200mg reagent, every experimenter 250mg reagent, every experimenter 300mg reagent, every experimenter 400mg reagent and every experimenter 500mg reagent.

The reagent that " gives (Administering) " can use in known the whole bag of tricks of those skilled in the art and the transmission system (delivery system) any one to implement or carry out.For example can be intravenous, per os, per nasal, intraperitoneal, through cerebrospinal fluid, through in implant, through mucous membrane, percutaneous, intramuscular, the blood vessel, in the intra-arterial, coronary artery, in the cardiac muscle or subcutaneous.

Term " PTEN-long analog (PTEN-long analogue) " is contained and is had one or more through modified amino acid; But at least 90% sequence similarity of reservation and SEQ ID NO:1, and as measure the active polypeptide of reservation or improvement inhibition tumor growth through in vivo (in vivo) institute hereinafter described.Clearly get rid of PTEN itself polypeptide of the residue 174-576 definition of SEQ ID NO:1 only (promptly by).

Term " PTEN-long variant (PTEN-long variant) " is encompassed in the N end of PTEN-long or C holds or both have one or more other aminoacid (have usually and be less than 5 other aminoacid) and keep the active polypeptide that perhaps improvement suppresses tumor growth as measuring through in vivo institute hereinafter described.

Term " PTEN-long analog variant (PTEN-long analogue variant) " is encompassed in the N end of PTEN-long analog (being SEQ ID NO:5) or C holds or both have one or more other aminoacid (have usually and be less than 5 other aminoacid) and keep the active polypeptide that perhaps improvement suppresses tumor growth as measuring through in vivo institute hereinafter described.

All embodiment that mention the PTEN-long analog among this paper are applicable to the PTEN-long variant under in addition necessary change.

PTEN-long is called PTEN-beta, PTEN-β, PTEN-S sometimes in addition.

The injectable drug transmission system that is used for PTEN-long, PTEN-long analog, PTEN-long variant, PTEN-long analog variant or its jointer (conjugate) separately comprises solution, suspension, gel, microsphere and polymerization injectable liquid (polymeric injectable); And can comprise excipient, change agent (solubility-altering agent) (for example ethanol, propylene glycol and sucrose) and polymer (for example gathering caprylyl acetone (polycaprylactone) and PLGA class) such as dissolubility.Implantable system (Implantable system) comprises club and pan, and can contain excipient, such as limiting examples PLGA with gather caprylyl acetone.

The oral delivery system (Oral delivery system) that is used for compositions of the present invention and chemical compound comprises tablet and capsule.These systems can contain excipient, such as binding agent (for example hydroxypropyl emthylcellulose, polyvinylpyrrolidone (polyvinyl pyrrolidone), other cellulosic material and starch), diluent (for example lactose and other sugar, starch, dicalcium phosphate and cellulosic material), disintegrating agent (for example starch polymer and cellulosic material) and lubricant (for example stearate and Pulvis Talci).

The through mucous membrane transmission system (Transmucosal delivery system) that is used for compositions of the present invention and chemical compound comprises paster, tablet, suppository, pessulum, gel and emulsifiable paste; And can contain excipient; Such as solubilizing agent and reinforcing agent (for example propylene glycol, bile salts and aminoacid) and other mediator (for example Polyethylene Glycol, fatty acid ester and derivant; And hydrophilic polymer, such as hydroxypropyl emthylcellulose and hyaluronic acid).

The transdermal delivery system (Dermal delivery system) that is used for compositions of the present invention and chemical compound comprises for example aqueous and non-aqueous gel, emulsifiable paste, multiple emulsion, microemulsion, liposome, ointment, aqueous and non-aqueous solution, lotion, aerosol, hydrocarbon base and powder; And can contain excipient, such as solubilizing agent, penetration enhancers (for example fatty acid, fatty acid ester, aliphatic alcohol and aminoacid) and hydrophilic polymer (for example polycarbophil (polycarbophil) and polyvinylpyrrolidone (polyvinylpyrrolidone)).In one embodiment, pharmaceutically acceptable supporting agent is liposome or transdermal enhancer.

The solution, suspension and the powder that are used for restored concerning the transmission system (reconstitutable delivery system) of compositions of the present invention and chemical compound comprise mediator, such as suspending agent (for example natural gum, Hydrargyri Oxydum Rubrum glue (zanthans), cellulose and sugar), wetting agent (for example Sorbitol), solubilizing agent (for example ethanol, water, PEG and propylene glycol), surfactant (for example sodium lauryl sulfate, this dish (Spans), tween (Tweens) and cetyl pyridinium), antiseptic and antioxidant (for example p-Hydroxybenzoate (paraben), vitamin E and C and ascorbic acid), anti-caking agent, smears and chelating agen (for example EDTA).

As used herein, " 5 ' ATR " is 5 ' substituting translation area described in the hereinafter experimental section.

As used herein, " pharmaceutical carriers (pharmaceutical carrier) " is pharmaceutically acceptable solvent, suspending agent or the mediator that is used for transmitting to animal or human's class The compounds of this invention or compositions.Supporting agent can be liquid, aerosol, gel or solid and selects according to the plan administering mode of being considered.

The meaning of " non-peptide reagent " is any chemical entities, includes, but is not limited to saccharic acid polymer (glycomer), polymer, micromolecule (promptly based on the molecule of hydrocarbon or molecular weight less than 1000 organic molecule), lipid, liposome.The instance of non-peptide reagent includes, but is not limited to PEG, Fmoc and FMS.

As used herein, " entity tumor " comprises carcinous and non-carcinous entity tumor.Carcinous entity tumor includes, but is not limited to cancer of bile ducts; The brain cancer comprises glioblastoma multiforme and medulloblastoma (medulloblastoma); Breast carcinoma; Cervical cancer; Choriocarcinoma; Colon cancer; Carcinoma of endometrium; Esophageal carcinoma; Gastric cancer; Last Intradermal vegetation (intraepithelial neoplasm) comprises Bao winton's disease (Bowen ' s disease) and Paget (Paget ' s disease); Hepatocarcinoma; Pulmonary carcinoma; Lymphoma comprises lymphogranulomatosis (Hodgkin ' s disease) and lymphocytic lymphoma; Neuroblastoma; Oral cancer comprises squamous cell carcinoma; Ovarian cancer comprises the ovarian cancer that is caused by epithelial cell, stromal cell, sexual cell and mesenchymal cell; Cancer of pancreas; Carcinoma of prostate; Colorectal carcinoma; Sarcoma comprises leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; Skin carcinoma comprises melanoma, card fort sarcoma (Kaposi ' s sarcoma), basal cell carcinoma (basocellular cancer) and squamous cell carcinoma; Carcinoma of testis comprises blastocyte tumor (spermocytoma, nonseminoma [teratoma, choriocarcinoma]), stromal tumors and germ cell tumor; Thyroid carcinoma comprises thyroid adenocarcinoma and medullary carcinoma; And renal carcinoma, comprise adenocarcinoma and Weir Mu Shi tumor (Wilms tumor), but do not comprise non-solid tissue tumor, such as leukemia and other hematology's vegetation, comprise acute lymphoblastic and myelomatosis; Multiple myeloma; Relevant leukemia of AIDS and adult T-cell leukemia-lymphoma.

The SEQ ID NO:1 of sequence table is a total length PTEN-long protein sequence.Among amino acid residue 1 to the 173 expression PTEN-long of SEQ ID NO:1 by the novel residue (referring to the description of following SEQ ID NO:2) of 5 ' ATR mRNA sequential coding.The classical initial methionine of PTEN is the amino acid residue 174 of SEQ ID NO:1.

SEQ ID NO:2 is a total length PTEN-long mRNA sequence.5 ' ATR sequence of PTEN-long starts from the nucleotide 513 (non-classical CUG start codon) of SEQ ID NO:2 and ends at the nucleotide 1031 of SEQ ID NO:2.The classical AUG start codon at 5 ' ATR and nucleotide 1032 places that start from SEQ ID NO:2 and end at the same frame of PTEN ORFs (open reading frame) of the nucleotide 2243 of SEQ ID NO:2.Therefore, the PTEN-long ORFs extends to the nucleotide 2243 of SEQ ID NO:2 and causes the N end of PTEN to increase by 173 aminoacid from the nucleotide 513 of SEQ ID NO:2.Said protein is called as PTEN-long.

SEQ ID NO:3 is the cDNA corresponding to total length PTEN-long mRNA sequence.

SEQ ID NO:4 is the peptide of PTEN-long protein sequence that comprises the amino acid residue 153 to 173 of SEQ ID NO:1.This unique peptide representative is not present in the unique epitope that derives from PTEN-long among the PTEN.

SEQ ID NO:5 is a total length PTEN-long analog protein sequence.The amino acid residue 1 of PTEN-long becomes Met to increase protein output from Leu in analog.

SEQ ID NO:6 is the total length PTEN-long analog mRNA sequence through modifying.5 ' ATR sequence of PTEN-long analog starts from the nucleotide 513 (through engineered AUG start codon) of SEQ ID NO:6 and ends at the nucleotide 1031 of SEQ ID NO:6.

SEQ ID NO:7 is the cDNA through modifying corresponding to the total length PTEN-long analog mRNA sequence of warp modification.

When providing scope in the description, should be appreciated that said scope comprises all integers and 0.1 unit and its any subrange in the said scope.For instance; 77% to 90% scope comprises 77.0%, 77.1%, 77.2%, 77.3%, 77.4%, 77.5%, 77.6%, 77.7%, 77.8%, 77.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9% and 90.0%, and scope 80% to 81.5% etc.

All combinations of various key elements as herein described all within the scope of the invention.

The present invention will be through obtaining fully to understand with reference to subsequently experiment details, but it will be apparent to those skilled in the art that the concrete experiment of being detailed only explains the present invention, and the present invention will be hereinafter more fully describes in subsequently claims.

The experiment details

The PTEN TIF is one of gene that the most often changes in the cancer.It serves as phosphatidylinositols 3,4, and the lipid phosphatase of 5-triphosphoric acid, and phosphatidylinositols 3,4,5-triphosphoric acid suppress the carcinogenic signal conduction from phosphatidyl-inositol 3-kinase (PI3K) and Akt again.The inspection that PTEN mRNA is carried out discloses the 5 ' untranslated region (UTR) and the same frame of PTEN ORFs (ORF) of 770bp.In this UTR ORF, there is substituting CUG start codon in 513 base pair places at classical AUG upstream from start codon in weak V (Kozak) sequence.Though the expression of classical PTEN ORF produces the protein of moving to about 55kDa, the expression that contains the PTEN cDNA of 5 ' UTR can produce 70kDa second protein that is called PTEN-long.The sudden change of initiation site shows that 55kDa PTEN produces from the translation that classical start codon begins, and PTEN-long is that substituting initiation site is initial from the upper reaches.The endogenous of immunoblotting explanation PTEN-long in various kinds of cell system that uses different PTEN antibody to carry out exists.The blocking gene that in ES cells, carries out is expressed and gene knockout research confirmation is this is actually PTEN than larger protein.The N end protein matter sequential coding signal peptide and the cracking site that are added show that PTEN-long gets into the secretion path.The preferential binding lectin concanavalin A of PTEN-long, this explains that it is by glycosylation.In addition, can pass through affinity purification, use to the antibody of PTEN and Heparan sulfate from conditioned medium purification PTEN-long.PTEN-long was also to the sensitivity of degrading during protease protection was in vivo analyzed, and normal PTEN not can, this shows that PTEN-long is positioned the cell membrane outside.

Reagent, cell line and antibody-E.C. 3.4.21.64 and Con A Concanavalin-A are available from sigma company (Sigma) (St. Louis, the Missouri State (St.Louis, MO)).Heparin-agarose and HiTrap heparin HP post are available from peace agate West Asia company (Amersham) (New Jersey skin SIKA tower (Piscataway, NJ)).Antibody to PTEN is available from the logical company (Cell Signaling) (Massachusetts Dan Fusi (Danvers MA)) of Menaphtame and Cascade Corp. (Cascade) (Winchester (Winchester MA), east, Massachusetts).Akt antibody is to be available from A Pusidetemilibo (Upstate Millipore) (card (Billerica, Ma)) in the Bill of Massachusetts available from logical company (Cell Signaling) (Massachusetts Dan Fusi (Danvers MA)) of Menaphtame and E cadherins antibody (E cadhein antibody).Through Yi Mude laboratory (Zymed Laboratories) (the polyclonal antibody that San Francisco, south, California (South San Fransisco, CA)) obtains through affinity purification to being seen epitope PRHQQLLPSLSSFFFSHRLPD (SEQ ID NO:3) in the novelty translation of PTEN.Secondary antibody is available from Pierre Si company (Pierce) (Illinois, USA Rockford (Rockford, IL)).HEK293, ZR-75-1, SKBR-3, MDAMB-361, BT549 and PC3 are available from US mode culture collection center (ATCC) (Manassas, Virginia (Manassas, VA)) and cultivate according to the guide that is provided.

Plasmid with construct body-like previous report; Through producing the encode complete ORFs of PTEN and the pCEP4-PTEN (Lee (Li), Simpson people 1998 such as (Simpson)) of 5 ' untranslated region in the NotI site of PTEN cDNA (being preserved in NCBI with U90351) being cloned into pCEP4 (hero company).Further connect adapter (adaptor) coded sequence and on this plasmid, prolong 5 ' UTR through the upper reaches at the original NotI restriction site (restriction site) that is used to clone.Adapter coding be positioned at classical initiation site-513 first maybe substituting CTG upstream from start codon 10 base pairs at the most.Suppose that adapter that substituting initiation site (putative alternate start site) sports ATG also can be used for producing second group of will express microscler formula effectively and expresses and construct body.In addition, classical start codon sudden change is brought out for ATA has also taken place, and body (Fig. 5 .1) is constructed in generation 4 kinds of differences altogether.Also with these 4 kinds variations and the ORFs sub-clone of original PTEN to MSCV (clone technology company (Clontech); In Mountain View, California (Mountainview, the CA)) retrovirus vector (retrovirus vector) to carry out stably express through infection.

Protease protection analyzes-with the HEK293 cell harvesting in the ice-cold PBS of no trypsin and with 5 * 10 ⁵Individual cell aliquot was cultivated 30 minutes with the E.C. 3.4.21.64 of 0.5 μ g/ml to 10 μ g/ml progressive concentration.Comprise that the contrast of using 0.1%Triton is with the proteinic ability of checking E.C. 3.4.21.64 degraded appointment.With 5mM PMSF stopped reaction.Dissolved cell and carry out immunoblotting in 2 * La Muli sample buffer (125nM Tris (pH 6.8), 20% glycerol, 0.05% bromophenol blue, 4%SDS, 10%2-mercaptoethanol) to PTEN, Akt and E cadherins.

Will be from mouse liver purification PTEN-from liver quick-freezing in liquid nitrogen of C57BL6 mice; Grind, and resuspending is in TNN buffer (50mM Tris (pH 7.4), 150mM NaCl, 0.5%NP-40,5mM EDTA, 3% glycerol, 1mM DTT, 1 * mammalian protease mixture inhibitor (Mammalian Protease Cocktail Inhibitor) [sigma company (Sigma)]).With mortar suspension is homogenized and under 4 degree 40, under the 000RPM centrifugal 1 hour.Use 0.45 micron and 0.22 micron filter filtering supernatant successively.Make propylene polydextran gel 200 size exclusion posts (peace agate West Asia company (Amersham)) pre-equilibration and apply sample with TNN, with after-applied buffer with the speed of 0.3ml/hr.Collect the 2ml fraction and compile the low-molecular-weight sample and put on the HiTrap heparin HP post (peace agate West Asia company (Amersham)) of pre-equilibration.Wash said post and progressively use 0.3M, 0.5M and the 1M NaCl TNN eluant solution protein of 3 times of column volumes with the TNN of 3 column volumes.Collect fraction and carry out immunoblotting with the 0.5ml increment to PTEN.

From culture medium purification PTEN heparin-make among the 10%FBS DMEM of HEK293 cell the 15cm culture dish to grow into fusion state (confluency).The DMEM that cell is not had a FBS with 15ml cultivates and spends the night.Collect the culture medium of 20 culture plates and pass through the filtration of 0.45 micron filter.Use AktaPrime (peace agate West Asia biotechnology company (AmershamBioscience)), under 4 ℃, use the flow velocity of 4ml/min, make 1ml heparin HP column equilibration with DMEM.Make conditioned medium pass through post subsequently with 1ml/min.BC200 (200mM NaCl, 50mMT Tris (pH 7.4), 1mM EDTA, 0.2%TritonX-100) column scrubber with 10 volumes.With 5ml 1M NaCl with 1ml/min with 1ml fraction elute protein.Measure the protein concentration of each fraction through the OD under the 280nm.Make half deposition of each fraction with 20% trichloroacetic acid, with the cold acetone washing, dry under vacuum.With 20 μ lLa Muli dissolving buffer restores protein and uses the antibody to PTEN and PTEN-long to carry out immunoblotting.

Is available from sigma company (Sigma) from serum purification PTEN-from the human serum of AB blood plasma.Filter 1ml serum and use a-protein/G agarose pre cleaning antibody to continue to cultivate 1 hour through 0.45 micron filter.Heparin-agarose (Heparin-agarose) is spent the night and washed with BC150 (150mM NaCl, 25mM Tris (pH 7.4), 1%NP-40,0.25% NaTDC, 1mM EDTA) in second day with cultivating together with the agarose contrast through the serum of pre cleaning.Carry out immunoblotting or only be directed against secondary antibody carrying out with La Muli sample buffer elute protein and to PTEN from the heavy chain pollution.

Concanavalin A catches-under subconfluence with BC500 (500mM NaCl, 20mM Tris (pH 7.4), 1%TritonX-100,1mM MnCl ₂, 1mM CaCl ₂, 1 * protease inhibitor cocktail) dissolving HEK293 cell.Centrifuge cell dissolves born of the same parents' thing and filters.Caught 1 hour with 20 microlitre concanavalin A agaroses (sigma company (Sigma)) down at 4 ℃.With the BC500 washing resin and with La Muli sample buffer elute protein.

The result

PTEN mRNA has the substituting initiation site in the upper reaches

Be preserved in PTEN mRNA (Lee (Li) and the grandson (Sun) 1997 of NCBI; Si Teke (Steck), Pei Sihaosi people 1997 such as (Pershouse)) contains 5 ' UTR widely.About 770bp continuous sequence in 5 ' UTR district and the same frame of start codon.The methionine of in this zone, not encoding; Yet, from several substituting initial CUG codons that come into existence apart from-519 of classical start codon.According to scansite (scansite.mit.edu) and prosite (www.ebi.ac.uk/ppsearch), the translation of this sequence discloses no identifiable domain.The translation in this whole zone is adding 173 aminoacid to PTEN, thereby makes its molecular mass be increased to about 70 kilodaltons (kilodaltons) (Fig. 2 and SEQ ID NO:1).

The comparison of the lineal congener of other PTEN discloses the translation sequences of homo sapiens UTR and can in the ORFs from the PTEN of various species, find.Chimpanzee, cattle, apis mellifera Linnaeus and Caenorhabditis elegans all contain and the homologous protein sequence of the translation product of homo sapiens 5 ' UTR (Fig. 3).In addition, the comparison of homo sapiens 5 ' UTR and house mouse PTEN 5 ' UTR shows extensive nucleotide homology (not shown).House mouse 5 ' the UTR of 522 base pairs compares highly homologous protein sequence (Fig. 5 .3) with classical start codon with frame translation and announcement with the translation of homo sapiens 5 ' UTR.The physical presence that derives from the aminoacid sequence of homo sapiens 5 ' UTR in the homology of 5 ' UTR and the translated protein of other species shows the evolution importance of this sequence.

PTEN mRNA can be from the initial translation of substituting upstream site.The overexpression of PTEN ORF produces the single protein band of 55kDa.Comprise 5 ' UTR can produce about 70kDa second than the larger protein band.Be present in many cell lines and different monoclonal antibody capable of using detects (Fig. 4) than larger protein band also endogenous in the PTEN immunoblotting.This also is present in the mice wild type ES cell than the larger protein band and is not present in the PTEN gene knockout mice ES cell, and in the mice ES of stably express PTEN shRNA clone, reduces (Fig. 4).Use the gene expression of the human pten protein in the siRNA blocking-up HEK293 cell also can cause the proteinic gene expression blocking-up of 70kDa.

The expression of plasmid in PTEN deletion form PC3 cell line of the ORF of coding PTEN causes producing 55kDa protein.When the plasmid overexpression of the 5 ' UTR that also encodes, produce 70kDa protein.Upper reaches supposition start codon sports ATG (Fig. 1, " ATG/ATG ") from CTG mainly makes immunoblotting pattern (immunoblot pattern) be transformed into 70kDa form (Fig. 4).Bring out through sudden change and to confirm that also the 55kDa band derives from ATG start codon (Fig. 4 " CTG/ATA ").

Therefore, 5 ' UTR is enough to the translation of the PTEN of initial more microscler formula.Therefore, 5 ' UTR be called 5 ' ATR (substituting translation area ( ALternately TRanslated RAnd detectedly be called " PTEN-long " egion)) than larger protein.

Confirm the generation of reorganization PTEN-long overexpression research through the polyclonal antibody of affinity purification and with it and the generation (Fig. 4) of endogenous form in the HEK293 cell to what the aminoacid from 5 ' ATR translation produced.Overexpression from molten born of the same parents' thing immunoblotting of the full cell of HEK293 cell and PC3 cell is studied; As if the PTEN-long that has various ways, this point out to exist possible post translational modification, Unrecorded splicing form or even 5 ' ATR in substituting start codon.

PTEN-long coding N end signal peptide

The N terminal sequence of PTEN-long contains a lot of alanine, and it possibly indicated and stride film sequence (transmembrane sequence) or signal peptide.Use SignalIP 3.0 to analyze translation sequences (translated sequence) and contain signal peptide (Fig. 5) with the said sequence of high probability degree (＞95%) prediction.Comprising to the signal peptide characteristic basic amino acid, is hydrophobicity section (hydrophobic stretch) subsequently.Suppose that the hydrophobicity transbilayer helix is by proline destruction and subsequently for having a little polar sequence.Also predict said sequence by cracking, show that protein should be discharged in the ER inner chamber.

PTEN is through glycosylation

One of sign of secreted and extracellular protein is in Golgi body (golgi apparatus), to add the glycoconjugates part, and this process is called glycosylation.Can add sugar on the agedoite among the common sequences N-X-S/T (X can not be proline) (Gu Puta (Gupta) and Bruner gram (Brunak) 2002) through the N-glycosylation; The hydroxyl of serine, threonine and tyrosine also can be the glycosylated target of so-called O-(Ju Leniusi (Julenius), More's lattice moral people 2005 such as (Molgaard)).PTEN has a plurality of O-glycosylation sites, but only has a N-glycosylation site.Whether the part that use agglutinin Con A Concanavalin-A (combination sugar moieties) confirms the PTEN complement in the HEK293 cell in catching analysis is through glycosylation.From these cell purifications to the PTEN mixture (Fig. 6) that contains about 50%PTEN-long.This explanation PTEN-long through the Cytoplasm 55kDa of glycosylation and PTEN form through glycosylation or PTEN-long cracking in the extracellular.

PTEN-long combines heparan and on cell surface, finds

PTEN combines multiple protein polysaccharide (proteoglycan); Such as syndecan (syndecan) and glypican (glypican), these Dan Baijutang come to light and are connected in cell membrane skin (outer leaflet of the membrane).These Dan Baijutang are both (Bolero (Blero) are opened people 2005 such as (Zhang)) in the highest extracellular molecules of heparinization degree.The previous PTEN that shown has affinity for the material of band height negative electricity, and this character of PTEN causes the high anionic property PIP3 of its preference (Da Si (Das), Dixon people 2003 such as (Dixon)).Because heparan is one of biomolecule with maximum negative electricity, so might heparan can mediate combining of PTEN and extracellular matrix.Use finds that from the protein extract of mouse liver PTEN combines heparan with high-affinity.In addition, use 1M NaCl from the heparin-agarose post continuously eluting PTEN also eluting obtain PTEN-long (Fig. 7).

The PTEN-long reply proteasome degradation that adheres to the cell membrane outer surface is responsive.In the protease protection is analyzed, thereby living cells is cultivated with protease and for protease, can not be seen through in order to protection all cells internal protein because of lipid film, so only extracellular protein degraded.Through shifting out the HEK293 cell and suspend from the adhere-wall culture thing with the E.C. 3.4.21.64 of progressive concentration with the PBS gentle agitation.With the PMSF stopped reaction and with La Muli buffer dissolved cell.PTEN-long is to E.C. 3.4.21.64 and E-cadherins (it is known extracellular protein) processes and displays sensitivity (Fig. 8).On the other hand, PTEN shows suitable protease sensitivity, and this 55kDa material that shows definite part is also in extracellular (because it is through glycosylation) or make and during Cytoplasm PTEN is exposed to the analysis of E.C. 3.4.21.64 some cytolysiss take place.Be exposed to E.C. 3.4.21.64 if comprise the contrast of using cell membrane permeability Triton with proof PTEN, so its degradable.Whether this is that the original form of PTEN or the cracking form of PTEN-long of moving to 55kDa and keeping the C end epitope of PTEN antibody are still waiting to observe.These data show that PTEN-long is positioned on the cell surface.

Solubility PTEN-long is secreted in the culture medium.

PTEN-long is present in that not get rid of a proteinic part on the cell surface solvable and be discharged into the probability in the cellular environment.Use heparin-agarose affinity purification PTEN-long from the serum-free condition culture medium of HEK293 cell.The post eluting discloses and has the PTEN (Fig. 9) of moving to the 50kDa molecular weight in the culture medium.The immunoblotting that uses the PTEN-long specific antibody to carry out has disclosed identical 50kDa material, shows that this protein keeps the sequence of coming from substituting initiation site translation and from the sequence of the C end epitope of PTEN monoclonal antibody.This hints that consumingly observing is that the PTEN part of 55kDa is in fact by the cracked translation product that derives from upper reaches start codon.

Further confirm that through overexpression PTEN-long in the HEK293 cell of constructing the body transfection through ATG/ATG PTEN is secreted in the culture medium.Using these cells to produce serum-free condition culture medium spends the night and uses PTEN monoclonal antibody 6H2.1 to make PTEN immunoprecipitation from the 1ml culture medium.Successfully from culture medium, make big PTEN band and less 55kDa band immunoprecipitation.Because the protein overexpression, so may not carry out proteinic suitable processing, this causes secreting the 70kDa PTEN of full-size.

PTEN finds in human serum.

One of the best source of physiology secreted material is a serum.Use heparin-agarose affinity purification PTEN from human serum.Particle matter is removed in centrifugal human serum and filtration.Subsequently with BC150 by 1: 5 the dilution and with a-protein/G widely pre cleaning with the removal IgG.Serum is cultivated with a small amount of heparin-agarose in batches.With La Muli buffer solution elution heparin-agarose and to PTEN eluent is carried out the trace reaction and only carries out polluting to eliminate heavy chain to secondary antibody.PTEN and PTEN-long find (Figure 10) in human serum.

The anti-angiogenesis activity of PTEN-long

The blood vessel formation against function of PTEN-long proves through following: (1) PTEN-long is weak expression in developmental mice retina usually, but in experience is degenerated new life's the growth blood vessel of (involution)/cell death visible high level expression (Figure 11); (2) find in the apoptosis blood vessel of PTEN-long in tumor.In addition, use from the partially purified PTEN-long of transfectional cell and handle epithelial cell, suppress cell migration and cell death inducing (Figure 12).In U87 in culture, HUVEC endotheliocyte or 293 cells, purified PTEN-long also can induce the cell death relevant with the apoptosis activation, as measured through Casprotease-3 cracking.

In vivo antitumor and the anti-angiogenesis activity of PTEN-long

Figure 13 demonstration is handled mice (A) with PTEN-long.Form xenograft with 2 positions (left side and right) for mice (n=5) injection glioblastoma cell line U87 in mammary fat pad.After the tumour transplatation, do not inject (w/PTEN-long) directly for a tumor injection PTEN-long and offside tumor.Derive from preparation also for the matched group injection (empty carrier) of one group of 5 mice with the simulation protein purification of empty carrier cells transfected.The offside tumor is not injected (w/ empty carrier) once more.Handled mice at 1-11 days and 13-14 days.Measure maximum gauge (cm) at appointed day with caliper.When gross tumor volume reach >=during 1cm, put to death mice.(B) pass through the transfection of PTEN-long expression vector in 293 cells, and use the affine resin of V5 to carry out partial purification, prepare protein with V5 peptide eluting subsequently.Figure 14 shows the survival branch rate (in the sky) with contrast and 14 days mice of PTEN-long injection processing.

Retina dyeing

Dyeing to PTEN-long in the p7 muroid retina and blood vessel discloses the PTEN-long selectively staining, at the moment that the muroid retinal vessel is grown, the hyaloid blood vessel that begins to degenerate.Antibody to PTEN-long is to epitope: N-PRHQQLLPSLSSFFFSHRLPD-C (SEQ ID NO:3).Use BS1-agglutinin (BS1-lectin) to carry out blood vessel dyeing.

Purification

In the method for a kind of purification PTEN-long, with ATG/ATG PTEN-long rotaring redyeing 293 cell and make cell dissolve born of the same parents' thing to pass through Ni ⁺Affinity column.All the time use Ni ⁺Post on the AKTA purifier, uses the imidazoles elution buffer to come purification PTEN-long.

Tumour regression

Before the injection through the xenograft of the U87 cell of PTEN (403 aminoacid of orf) or PTEN-long transfection.Injected back 7 days, and compared with PTEN overexpression group (among the n=4 4), the breast blood vessel reduces in PTEN-long overexpression group.This shows that PTEN-long can influence tumor environment.

Discuss

In the PTEN immunoblotting of molten born of the same parents' thing of cell and tissue, fitly observe second than the larger protein band.Confirmation is that the evidence of PTEN comprises than big band: detect than the larger protein band with different PTEN monoclonal antibodies; When handling cell or rejecting the PTEN locus (locus) of mice, do not exist than larger protein with siRNA to PTEN.Observe greater than the 5 ' UTR of the PTEN of 700 base pairs and the same frame of classical start codon of PTEN.In addition, there is the CUG codon at 522 base pair places at the PTEN upper reaches, if its translation, the size in the so soluble PTEN immunoblotting than the larger protein band.Although itself and strong V sequence (Kozak sequence) have nothing to do its reservation-1 cytosine and+1 guanosine sequence.Serve as interpreter and when adding on the PTEN ORF, should form the protein of about 70kDa, it is the molecular mass of viewed big PTEN band.

The translation of this sequence is present in the actual coding sequence of the lineal congener of many PTEN.Mice 5 ' UTR also is examined, and has similar band because observe in the molten born of the same parents' thing of mouse tissue.Mice 5 ' UTR nucleotide sequence and homo sapiens 5 ' UTR height homology and similarly with the same frame of start codon.Two maybe substituting start codon be present in-522 and disclose aminoacid sequence and homo sapiens's sequence 90%+ homology with-516 places and this sequence from the translation in those sites.The conservative of this putative protein matter is significant and has shown the evolution importance of this sequence.In order to describe this sequence preferably, its 5 ' ATR or substituting translation area that is renamed as PTEN possibly translated to describe it.

Construct plasmid as follows: wherein, the ORFs of PTEN clone with 5 ' ATR and separately with the expression of this reorganization PTEN with produce 403 amino acid whose classical ORFs and compare.The expression plasmid (expression plasmid) of the classical ORF that only contains PTEN of moving to the single band of 55kDa with generation is compared, and comprises that 5 ' ATR can produce the second higher pten protein band of moving to about 70kDa.Only account for the sub-fraction of the gross protein of translating than larger protein; Yet, suppose that initiation site sports ATG and makes protein rate turn to be mainly big form.

The conservative of the protein sequence that obtains from 5 ' ATR has shown that it has exceeded the evolution product.The N end contains one section aliphatic amino acid, predicts that it is for striding the film sequence.Using the N end of Prosite and Signal 3.0IP prediction PTEN-long is signal peptide, and being next to is thereafter protease cracking site (protease cleavage site).

Using in vivo, whether protease protection analytical test PTEN-long is positioned on the cell outer surface of cell.PTEN-long shows that the amount with extracellular protease increases and degraded gradually, PTEN then not can, this show at least some PTEN-long in the extracellular and at least part be connected in the cell membrane skin.This is that the active lipid phosphatase of hint is positioned at the result who induces one to become interested most on the cell membrane skin.Two families of cell membrane conjunction type Dan Baijutang, glypican and syndecan outside before in the pten protein complex, identifying.

PTEN is present in the probability of not getting rid of solubility secreted PTEN on the cell surface.Syndecan and glypican are both in the highest Dan Baijutang of heparinization degree.Heparan is the glucosaminoglycan of band height negative electricity and has shown that PTEN has affinity for anion that this part has explained the reason of the PIP3 of zone of preference height negative electricity as its substrate.Can use the heparin-agarose post to carry out purification from optimization experiment announcement PTEN and the PTEN-long of mouse liver purification PTEN.In addition, use the heparin-agarose post, obtain the protein of about 50kDa from the serum-free condition culture medium purification of HEK293 cell.Purified protein is had specific monoclonal antibody and is discerned to the polyclonal antibody of the unique amino acid residue that exists among the PTEN-long to PTEN.Previous PTEN-long antibody is only discerned the protein band of about 70kDa.The observed result that two kinds of antibody can be discerned a band shows, and Proteolytic enzyme processing (proteolytic processing) possibly take place, and is the fragment that keeps the epitope of two kinds of antibody among the PTEN through the observed protein of immunoblotting.PTEN and PTEN-long also can use the heparin affinity purification from human serum purification.

10 years in the past, accumulated the lot of documents of imagining the PTEN sequence.There is the PTEN of novel form in this paper proof, and it is translated and be secreted into skin and the ECS from substituting site.

In vivo the result shows that PTEN-long is novel antitumoral compounds, and it is present in the human serum usually and has angiogenesis inhibitor and short apoptosis character.

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Claims

1. a separated human phosphatase enzyme and the long polypeptide of tensin congener (phosphatase and tensin homolog long polypeptide who comprises continuous amino acid residue with sequence shown in the SEQ ID NO:1; PTEN-long); Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5 or its variant separately.

2. a peptide species is characterized in that, comprises the residue 1 to 173 of (i) SEQ ID NO:1, or (ii) comprises its analog of the residue 1 to 173 of SEQ ID NO:5, or the (iii) residue 22 to 516 of SEQ ID NO:1.

3. pharmaceutical composition; It is characterized in that; Contain the phosphatase and the long polypeptide of tensin congener (PTEN-long) that comprise the continuous amino acid residue; Said amino acid residue has sequence shown in the SEQ ID NO:1, or comprises its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprises its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5.

4. method of treating experimenter's entity tumor; It is characterized in that; Comprise and give said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprise with continuous amino acid residue of sequence shown in the SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with effective treatment experimenter's entity tumor.

5. the method for the growth of an entity tumor that suppresses the experimenter; It is characterized in that; Comprise and give said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprise with continuous amino acid residue of sequence shown in the SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with effective growth that suppresses experimenter's entity tumor.

6. the method for the angiogenesis in the entity tumor that suppresses the experimenter; It is characterized in that; Comprise and give said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprise with continuous amino acid residue of sequence shown in the SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with the angiogenesis in effective inhibition experimenter's the entity tumor.

7. apoptotic method takes place in the blood vessel epithelial cell of the blood vessel in the entity tumor of inducing the experimenter; It is characterized in that; Comprise and give said experimenter a certain amount of human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprise with continuous amino acid residue of sequence shown in the SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog with continuous amino acid residue of sequence shown in the SEQ ID NO:5, with the blood vessel epithelial cell generation apoptosis of the blood vessel in the entity tumor of effectively inducing the experimenter.

8. according to claim 4,5,6 or 7 described methods, it is characterized in that said tumor is a cancerous tumour.

9. method according to claim 8 is characterized in that, said cancerous tumour is the tumor of experimenter's neurogliocyte, prostate, ovary, uterus, endometrium, breast, melanocyte, kidney, lung, colon, head, cervical region or pancreas.

10. method according to claim 8 is characterized in that, said cancerous tumour is by PTEN or by the activation of PI3K path or be the PTEN feminine gender.

11., it is characterized in that said PTEN-long or said its analog or said PTEN-long fragment are through giving said experimenter in the said entity tumor of direct introducing according to the described method of arbitrary claim in the claim 4 to 10.

12. method according to claim 11 is characterized in that, said PTEN-long or said its analog or said PTEN-long fragment are to be injected in the said entity tumor.

13. method according to claim 11 is characterized in that, said PTEN-long or said its analog or said PTEN-long fragment are directly to introduce in the said entity tumor through conduit.

14., it is characterized in that said PTEN-long or said its analog or said PTEN-long fragment are to supply in the blood vessel of said entity tumor through direct introducing to give said experimenter according to the described method of arbitrary claim in the claim 4 to 10.

15. method according to claim 14 is characterized in that, said PTEN-long or said its analog or said PTEN-long fragment are to be injected in the said blood vessel of the said entity tumor of supply.

16. method according to claim 14 is characterized in that, said PTEN-long or said its analog or said PTEN-long fragment are directly to introduce in the said blood vessel of the said entity tumor of supply through conduit.

17., it is characterized in that said PTEN-long or said its analog or said PTEN-long fragment are that intravenous gives said experimenter according to the described method of arbitrary claim in the claim 4 to 10.

18., it is characterized in that said PTEN-long or said its analog or said PTEN-long fragment are the subcutaneous said experimenters that gives according to the described method of arbitrary claim in the claim 4 to 10.

19. method of treating experimenter's entity tumor; It is characterized in that; Comprise and give said experimenter a certain amount of expression vector; Said expression vector codes comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) of SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise its analog of SEQ ID NO:5, in the cell of said entity tumor, to express effectively PTEN-long, PTEN-long fragment or said its analog of the amount of the said experimenter's of treatment said entity tumor.

20. human phosphatase enzyme and long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprising its analog of SEQ ID NO:5, it is used to treat experimenter's entity tumor, suppresses the growth of experimenter's entity tumor; Induce the blood vessel epithelial cell generation apoptosis in experimenter's the entity tumor, or suppress the angiogenesis in experimenter's the entity tumor.

21. PTEN-long according to claim 20 or its fragment or said its analog is characterized in that said tumor is a cancerous tumour.

22. PTEN-long according to claim 21 or its fragment or its analog is characterized in that, said cancerous tumour is neuroglia cancer, carcinoma of prostate, mastocarcinoma, carcinoma of endometrium, melanocarcinoma, renal carcinoma or pulmonary carcinoma tumor.

23. PTEN-long according to claim 21 or its fragment or said its analog is characterized in that, said cancerous tumour is by PTEN or by the activation of PI3K path or be the PTEN feminine gender.

24. compositions; It is characterized in that; The human phosphatase enzyme that comprises SEQ ID NO:1 and the long polypeptide of tensin congener (PTEN-long) that comprise the non-peptide reagent of bonding; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise its analog of SEQ ID NO:5, said non-peptide reagent increases the plasma half-life of said PTEN-long, PTEN-long fragment or said its analog respectively.

25. compositions according to claim 24 is characterized in that, further comprises pharmaceutical carriers.

26. compositions according to claim 24 is characterized in that, the said non-peptide reagent that increases said plasma half-life is Polyethylene Glycol (PEG).

27. compositions according to claim 26 is characterized in that, the C end of the said PTEN-long of said PEG bonding, PTEN-long fragment or said its analog or N end.

28. compositions according to claim 24 is characterized in that, the said non-peptide reagent that increases said plasma half-life is chloro-carbonic acid-9-fluorenyl methyl ester (Fmoc) or (7-sulfonic group)-9-fluorenylmethyloxycarbonyl.

29. separated nucleic acid; It is characterized in that; Coding comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) of SEQ ID NO:1, or its fragment of the residue 22 to 516 that comprises SEQ ID NO:1 of encoding, or encodes and comprise its analog of SEQ ID NO:5.

30. nucleic acid according to claim 29 is characterized in that, said nucleic acid comprises the continuous nucleotide with sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.

31. nucleic acid according to claim 30 is characterized in that, said nucleic acid comprises the continuous nucleotide residue 503 to 2243 of sequence shown in SEQ ID NO:2 or the SEQ ID NO:6.

32. nucleic acid according to claim 29 is characterized in that, said nucleic acid comprises the continuous nucleotide with sequence shown in SEQ ID NO:3 or the SEQ ID NO:7.

33. nucleic acid according to claim 32 is characterized in that, said nucleic acid comprises the continuous nucleotide residue 503 to 2243 of sequence shown in SEQ ID NO:3 or the SEQ ID NO:7.

34. nucleic acid according to claim 29 is characterized in that, said nucleic acid is RNA.

35. nucleic acid according to claim 33 is characterized in that, said nucleic acid is DNA.

36. nucleic acid according to claim 35 is characterized in that, said nucleic acid is cDNA.

37. expression vector; It is characterized in that; Comprise a kind of nucleic acid; Said nucleic acid coding comprises human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) of SEQ ID NO:1, or comprises its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprises its analog of SEQ ID NO:5.

38. transformant; It can express human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog of SEQ ID NO:5, it is characterized in that will encode the recombinant DNA of PTEN-long or its analog of said cell is incorporated in its genome.

39. host cell; It can express human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that comprises SEQ ID NO:1; Or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1; Or comprise its analog of SEQ ID NO:5, it is characterized in that said host cell comprises the plasmid of coding PTEN-long or its analog.

40., it is characterized in that said host cell is a bacterial cell according to the described host cell of claim 39.

41., it is characterized in that said host cell is a mammalian cell according to the described host cell of claim 39.

42. method; It is characterized in that; Comprise human phosphatase enzyme and the long polypeptide of tensin congener (PTEN-long) that mixing (1) comprises SEQ ID NO:1, or comprise its fragment of the residue 22 to 516 of SEQ ID NO:1, or comprise its analog of SEQ ID NO:5; (2) the non-peptide reagent of the plasma half-life of increase PTEN-long or said its analog; So that comprise the PTEN-long of SEQ ID NO:1 or comprise SEQ ID NO:1 residue 22 to 516 said its fragment or comprise said its analogue bonded said non-peptide reagent of SEQ ID NO:5, said non-peptide reagent increases the said plasma half-life of said PTEN-long, PTEN-long fragment or said its analog respectively.

43. separated nucleic acid molecules; It is characterized in that, form with the complementary series of the said nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:6 or with its complementary series of SEQ ID NO:2 or SEQ ID NO:6 specific hybrid respectively by the fragment with at least 20 nucleotide of SEQ ID NO:2 or SEQ ID NO:6 nucleotide sequence or under stringent condition.

44. a separated antibody or its fragment is characterized in that, said antibody or its fragment combine (1) to comprise the PTEN-long polypeptide of the amino acid residue 1-173 of SEQ ID NO:1; Or (2) comprise its analog of SEQ ID NO:5; Or (3) have the aminoacid of sequence shown in the SEQ ID NO:3; Or (4) comprise the PTEN-long fragment of the residue 22 to 516 of SEQ ID NO:1.

45., it is characterized in that said antibody is monoclonal antibody according to the described separated antibody of claim 44.

46., it is characterized in that said antibody is antibody fragment according to the described separated antibody of claim 44.

47., it is characterized in that said antibody fragment is Fab, Fab ', F (ab ') according to the described antibody fragment of claim 46 ₂Or Fv fragment.

48., it is characterized in that said antibody fragment is a single-chain antibody according to the described antibody of claim 47.

49., it is characterized in that said antibody is humanized antibody according to the described antibody of claim 44.

50. a separated antibody or its fragment; It is characterized in that; Said antibody or its fragment combine the conformation epitope of PTEN-long, and said PTEN-long comprises the sequence shown in the residue 1-173 of SEQ ID NO:1, but said antibody does not combine PTEN.

51., it is characterized in that the epitope that said antibodies is made up of the residue 153-173 of arbitrary SEQ ID NO:1 according to the described antibody of claim 50.

52. a separated antibody or its fragment is characterized in that, said antibody or its fragment combine the epitope of PTEN-long, and said PTEN-long comprises the sequence shown in the residue 1-173 of SEQ ID NO:1, but said antibody does not combine PTEN.

53., it is characterized in that the residue 153-173 of said antibodies SEQ ID NO:1 or its part according to the described antibody of claim 52.

54. the fragments of peptides of SEQ ID NO:1 or SEQ ID NO:5 is characterized in that, it is active that said fragment has antitumor, angiogenesis inhibitor or anti-apoptotic.

55., it is characterized in that said peptide comprises 5-10,10-20 is individual, 20-30 is individual or 30-40 aminoacid according to the described peptide of claim 54.

56., it is characterized in that said peptide comprises the amino acid/11-173 of SEQ ID NO:1 or SEQ ID NO:5 according to the described peptide of claim 54.

57. according to the described peptide of claim 54, it is characterized in that, comprise the continuous amino acid residue 22 to 516 of SEQ ID NO:1.

58. according to claim 54 or 57 described peptides, it is characterized in that, do not comprise the continuous amino acid residue 174 to 576 of SEQ ID NO:1 or SEQ ID NO:5.